EP3474007A1 - Determination method, analysis method, and analysis system - Google Patents

Determination method, analysis method, and analysis system Download PDF

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Publication number
EP3474007A1
EP3474007A1 EP18201626.1A EP18201626A EP3474007A1 EP 3474007 A1 EP3474007 A1 EP 3474007A1 EP 18201626 A EP18201626 A EP 18201626A EP 3474007 A1 EP3474007 A1 EP 3474007A1
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EP
European Patent Office
Prior art keywords
solution
analyte
absorbance
flow path
value related
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Granted
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EP18201626.1A
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German (de)
French (fr)
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EP3474007B1 (en
Inventor
Takanari SHIGEMITSU
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Arkray Inc
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Arkray Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44769—Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells

Definitions

  • the present application relates to a determination method, an analysis method, and an analysis system.
  • specimen analysis by capillary electrophoresis has conventionally been conducted.
  • specimen analysis by electrophoresis using a microchip provided with a capillary flow path has been conducted in order to miniaturize and simplify an apparatus.
  • an analyzer for analyzing blood proteins such as hemoglobin by capillary electrophoresis has been proposed (see, for example, Patent Document 1).
  • An analysis method for analyzing HbA1c which is a glycated hemoglobin using an electrophoresis chip has also been proposed (see, for example, Patent Document 2).
  • a capillary In capillary electrophoresis, a capillary is irradiated with light, light transmitted through the capillary is detected, and a sample moving in the capillary is analyzed.
  • a sample moving in the capillary is analyzed.
  • glycated hemoglobin can be measured using a rapid and inexpensive microchip.
  • stray light in optical detection exerts a large influence on the measurement accuracy, and a function of removing stray light is provided in an analyzer, the configuration of the analyzer becomes complicated and expensive.
  • the microchip described in Patent Document 3 has a function of reducing the influence of stray light, which enables analysis at a low cost with a simple device, in some cases, an influence of stray light is not reduced due to an optical fault of the analyzer, a production defect such as a scratch in a mechanism for removing stray light of a microchip, or foreign matter contamination in a mechanism for removing stray light of a microchip.
  • At least preferred embodiments of the present invention may provide a determination method, an analysis method, and an analysis system that can achieve high measurement accuracy easily and inexpensively.
  • a first aspect of the invention provides a determination method for determining whether the state of optical detection of a microchip is favorable or poor, comprising:
  • At least one of the first solution or the second solution contains a specific substance
  • the component difference between the first solution and the second solution is a difference in concentration of the specific substance between the first solution and the second solution.
  • the specific substance is an electrically neutral substance.
  • the specific substance may be at least one of 1-(3-sulfopropyl) pyridinium hydroxide inner salt or polyoxyalkylene alkyl ether.
  • the optically detected value related to the component difference is an absorbance or an absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution, and in the determination process, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value is determined that the state of the optical detection is favorable, and a value less than the threshold value is determined that the state of the optical detection is poor.
  • a shear flow is generated at a connecting portion between the capillary flow path and the sample reservoir in the supply process.
  • An analysis method may comprise the determination method as set out above, and in the detection process, with respect to the separated component, a value related to the analyte is optically detected together with the value related to the component difference between the first solution and the second solution other than the value related to the analyte.
  • the value related to the analyte is corrected based on the optically detected value related to the component difference.
  • At least one of the first solution or the second solution contains a specific substance
  • the optically detected value related to the component difference is an absorbance or an absorbance change amount derived from a difference in concentration of the specific substance between the first solution and the second solution
  • the value related to the analyte is an absorbance or an absorbance change amount derived from the analyte
  • the absorbance or the absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution is compared with an absorbance or an absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or the absorbance change amount derived from the analyte is corrected according to the result of the comparison.
  • the analyte may be a biologically derived substance.
  • the microchip is not reused.
  • a second aspect of the invention provides an analysis system for determining whether the state of optical detection of a microchip is favorable or poor, comprising:
  • the value related to a component difference is an absorbance or an absorbance change amount derived from a difference in concentration of the specific substance between the first solution and the second solution
  • the value related to the analyte is an absorbance or an absorbance change amount derived from the analyte
  • an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value is determined that the state of the optical detection is favorable, and a value less than the threshold value is determined that the state of the optical detection is poor.
  • the determination means comprise correction means for correcting, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference, other than the value related to the analyte.
  • the determination means comprise correction means for correcting, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference, other than the value related to the analyte, and in the correction means, the absorbance or the absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution is compared with an absorbance or an absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or the absorbance change amount derived from the analyte is corrected according to the result of the comparison.
  • At least preferred embodiments of the present invention may provide a determination method, an analysis method, and an analysis system that can achieve high measurement accuracy easily and inexpensively can be provided.
  • the numerical range expressed by using “to” means a range including numerical values described before and after “to” as a lower limit value and an upper limit value.
  • a determination method includes: a supply process in which, by using a microchip including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side, the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte; a separation process in which, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path; a detection process for optically detecting a value related to a component difference between the first solution and the second solution (an optical value related to component difference) other than a value related to the analyte (an optical value related to the analyte) for the separated component; and a determination process for determining whether the state of the optical detection is favorable or poor by comparing the optically detected value related to the component difference with
  • a value related to a component difference between the first solution and the second solution other than a value related to an analyte is optically detected for the component separated in the capillary flow path by electrophoresis. Then, by comparing the optically detected value related to the component difference with a predetermined threshold value, whether the state of optical detection is favorable or poor is determined. As a result, it is possible to determine whether the state of optical detection of a microchip is favorable or poor, and it is possible to easily and inexpensively achieve high measurement accuracy even when a microchip including a capillary flow path is used.
  • a value for optically detected component difference is an absorbance or an absorbance change amount derived from a difference in concentration of a specific substance (excluding an analyte such as a biologically derived substance) contained in at least one of the first solution or the second solution
  • the above-described absorbance or absorbance change amount may be compared with a predetermined threshold value to determine whether the state of optical detection is favorable or poor.
  • the numerical values of an absorbance and absorbance change amount derived from a difference in concentration of a specific substance (excluding an analyte such as a biologically derived substance) contained in at least one of the first solution or the second solution tend to decrease as a stray light ratio increases, or the state of optical detection deteriorates.
  • results of the absorbance and absorbance change amount described above vary depending on the states of the microchips, for example, a production defect such as a scratch in a mechanism for removing stray light of a microchip, or a foreign matter contamination in a mechanism for removing stray light of a microchip.
  • an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value indicates that the state of optical detection is favorable (i.e. a value equal to or larger than the threshold value leads to the determination that the state of optical detection is favorable), and a value less than the threshold value indicates that the state of optical detection is poor (i.e. a value less than the threshold value leads to the determination that the state of optical detection is poor).
  • a value related to the component difference between the first solution and the second solution other than a value related to an analyte is optically detected, and whether the state of the optical detection is favorable or poor is determined by using a value related to such component difference.
  • the state of optical detection is favorable or poor by using a value related to an analyte
  • a microchip used in the determination method is used for a variety of analysis methods for a variety of samples, and is preferably used for analysis of substances (preferably biologically derived substances) in a sample by electrophoresis (preferably capillary electrophoresis).
  • samples include a specimen derived from a living organism, a specimen derived from the environment, a metal, a chemical substance, and a pharmaceutical.
  • the specimen derived from a living organism is not particularly limited, and examples thereof include urine, blood, hair, saliva, sweat, and nail.
  • the blood specimen include a red blood cell, whole blood, serum, and plasma.
  • Examples of the living body include a human, a non-human animal, and a plant, and examples of the non-human animal include a mammal other than humans, an amphibian reptile, a fish, a shellfish, and an insect.
  • the specimen derived from the environment is not particularly limited, and examples thereof include food, water, soil, atmosphere, and air. Examples of the food include a fresh food, and a processed food. Examples of the water include drinking water, groundwater, river water, sea water, and domestic wastewater.
  • the sample is preferably blood collected from a human body or the like.
  • As the analyte a biologically derived substance contained in a sample is preferable, and the biologically derived substance contained in blood is more preferable. Hemoglobin is also preferable among biological substances.
  • a diluted solution prepared, for example, by suspending, dispersing, or dissolving a solid including an analyte and a liquid containing an analyte (stock solution) in a liquid medium may be used as a sample.
  • the liquid medium include water, and a buffer solution.
  • Examples of an analyte included in blood include hemoglobin (Hb), albumin (Alb), globulin ( ⁇ 1, ⁇ 2, ⁇ , ⁇ globulin), and fibrinogen.
  • hemoglobin include normal hemoglobin (HbA0), glycated hemoglobin, modified hemoglobin, fetal hemoglobin (HbF), and mutated hemoglobin.
  • HbA1a hemoglobin Ala
  • HbA1b hemoglobin A1b
  • HbA1c hemoglobin A1c
  • GHbLys examples of an analyte included in blood include hemoglobin (Hb), albumin (Alb), globulin ( ⁇ 1, ⁇ 2, ⁇ , ⁇ globulin), and fibrinogen.
  • HbA0 normal hemoglobin
  • glycated hemoglobin include hemoglobin Ala (HbA1a), hemoglobin A1b (HbA1b), hemoglobin A1c (HbA1c), and
  • hemoglobin A1c examples include stable HbA1c (S-HbA1c), and unstable HbA1c (L-HbA1c).
  • modified hemoglobin examples include carbamylated Hb, and acetylated Hb.
  • mutated hemoglobin examples include hemoglobin C (HbC), hemoglobin D (HbD), hemoglobin E (HbE), and hemoglobin S (HbS).
  • At least one of the first solution or the second solution contains a specific substance
  • the component difference between the first solution and the second solution is preferably the difference in concentration of a specific substance between the first solution and the second solution.
  • the first solution and the second solution preferably contain a specific substance, and more preferably, only the second solution contains a specific substance.
  • the specific substance is preferably an electrically neutral substance.
  • the electrically neutral substance include a substance that is partially charged and is neutral as a whole, and a substance that does not have partial charges and is neutral, and specific examples thereof include an amphoteric substance and a nonionic substance.
  • the specific substance may be used singly or two or more kinds thereof may be used. When two or more kinds of the specific substances are used, all the specific substances may be contained in the first solution and the second solution, respectively, or at least one of the specific substances may be contained in only one of the first solution and the second solution.
  • amphoteric substance examples include phosphobetaine, sulfobetaine, and carbobetaine. More specifically, 1-(3-sulfopropyl) pyridinium hydroxide inner salt is preferable.
  • nonionic substance examples include a sugar such as glucose or starch, urea, an alcohol such as polyethylene glycol, a nonionic surfactant, and a nonionic polymer.
  • a sugar such as glucose or starch
  • urea an alcohol
  • polyethylene glycol such as polyethylene glycol
  • nonionic surfactant such as polyethylene glycol
  • nonionic polymer such as polyethylene glycol
  • polyoxyalkylene alkyl ether is preferable.
  • the second solution preferably contains an electrically neutral substance.
  • an electrically neutral substance contained in the second solution is subjected to an electroosmotic flow, and is electrophoresed at the interface between the first solution and the second solution in the capillary flow path, whereby whether the state of optical detection is favorable or poor is determined without being affected by measurement of an analyte.
  • Fig. 1 is a schematic configuration diagram of a microchip used in a determination method and an analysis method of one embodiment
  • Fig. 1A is a top view
  • Fig. 1B is a sectional view taken along line C'-C' of Fig 1A
  • a microchip 100 includes a sample reservoir 1, an electrophoresis liquid reservoir 3, a capillary flow path 2, and a detector 4.
  • the microchip 100 may be manufactured, for example, by joining a pair of base materials, which are substantially rectangular plate-shaped members.
  • the microchip 100 may be manufactured by joining a base material having through holes corresponding to the sample reservoir 1 and the electrophoresis liquid reservoir 3 and a base material including a fine groove corresponding to the capillary flow path 2 in such a manner that both ends of the groove face portions of the two through holes respectively.
  • Examples of the material of the base material constituting the microchip include glass, fused silica, and a resin, and from the viewpoints of cost, ease of processing, and ease of immobilization of a cationic polymer, a resin is preferable.
  • the resin include an acrylic resin such as polymethyl methacrylate (PMMA), polymethyl methacrylate, polycarbonate, polyvinylidene chloride, cyclic polyolefin, polyether ether ketone, polystyrene, polytetrafluoroethylene (PTFE), cycloolefin, polypropylene, and polyethylene, and from the viewpoint of excellent light transmittance, methyl polymethacrylate is preferable.
  • a disposable type analysis chip not for reusing may be used.
  • the sample reservoir 1 is a tank that is supplied with a second solution containing an analyte such as a biologically derived substance from an opening of the sample reservoir 1 and stores the supplied second solution.
  • the sample reservoir 1 is connected to the end of the capillary flow path 2.
  • the main agent of a diluent is not particularly limited, and examples thereof include water, and physiological saline, and a substance which can be contained in the first solution for electrophoresis described below as a preferred example may be added to the diluent.
  • the diluent may be one obtained by adding a compound including a cathodic group to the main agent.
  • the compound including a cathodic group include a cathodic group-containing polysaccharide, and more specific examples thereof include a sulfated polysaccharide, a carboxylated polysaccharide, a sulfonated polysaccharide, and a phosphorylated polysaccharide.
  • carboxylated polysaccharide alginic acid and a salt thereof (for example, sodium alginate) is preferable.
  • sulfated polysaccharide for example, chondroitin sulfate is preferable.
  • chondroitin sulfuric acid A, B, C, D, E, H, and K, and any of them may be used.
  • concentration of a compound including a cathodic group chondroitin sulfate
  • the electrophoresis liquid reservoir 3 is a reservoir for supplying a first solution for electrophoresis from an opening of the electrophoresis reservoir 3 and storing the supplied first solution.
  • the electrophoresis liquid reservoir 3 is connected to an end of the capillary flow path 2, and the first solution is filled in the capillary flow path 2 by pressurization.
  • the first solution which is an electrophoresis liquid used for electrophoresis is a medium which is supplied from the opening to the electrophoresis liquid reservoir 3 and filled in the capillary flow path 2 to generate electroosmotic flow in electrophoresis.
  • the first solution includes water, and/or physiological saline and the like.
  • the first solution preferably contains an acid. Examples of the acid include citric acid, maleic acid, tartaric acid, succinic acid, fumaric acid, phthalic acid, malonic acid, and malic acid.
  • the first solution preferably contains a weak base. Examples of the weak base include arginine, lysine, histidine, and a tris.
  • the pH of the first solution is preferably, for example, in the range of pH from 4.5 to 6.
  • the buffer of the first solution include MES, ADA, ACES, BES, MOPS, TES, and HEPES.
  • the compound including a cathodic group described in the description of the diluent may also be added to the first solution.
  • the concentration of the compound including a cathodic group is preferably, for example, in the range of from 0.01% by mass to 5% by mass.
  • the capillary flow path 2 is connected to the sample reservoir 1 and the electrophoresis liquid reservoir 3, and is a flow path for analyzing an analyte in the second solution supplied to the sample reservoir 1, and is preferably a capillary channel for analyzing an analyte by electrophoresis.
  • the size of the capillary flow path 2 is not particularly limited, and preferably, for example, the depth is from 25 ⁇ m to 100 ⁇ m, the width is from 25 ⁇ m to 100 ⁇ m, and the length is from 5 mm to 150 mm.
  • the capillary flow path 2 may be provided with an electric charge, for example, a positive electric charge on a wall face.
  • the method of imparting a positive charge is not particularly limited, and a cationic polymer may be immobilized in a region corresponding to the capillary flow path 2 in the above-described pair of base materials before joining the pair of base materials described above.
  • the detection place 4 is for entering and emitting light to be analyzed when conducting analysis by electrophoresis on the microchip 100, and is used, for example, for measuring absorbance.
  • the detection place 4 is formed at a position facing at least a portion of the capillary flow path 2.
  • the position of the detection place 4 may be appropriately determined according to the length of the capillary flow path 2 and the like.
  • the determination method includes a supply process in which, by using the microchip 100 described above, the capillary flow path 2 is filled with the first solution and the sample reservoir 1 is supplied with the second solution containing an analyte.
  • the first solution stored in the electrophoresis liquid reservoir 3 may be pressurized to fill the capillary flow path 2 with the first solution, and as the second solution, one obtained by diluting a sample containing an analyte with the aforementioned diluent may be used.
  • the determination method includes a separation process in which, by applying a voltage between the sample reservoir 1 supplied with the second solution and an inside of the capillary flow path 2 filled with the first solution, a component contained in the second solution moves in the capillary flow path 2 and the aforementioned component is separated in the capillary flow path 2.
  • the electrophoresis liquid reservoir 3 has been supplied with the first solution, and the capillary flow path 2 has been filled with the first solution by pressurization, and the sample reservoir 1 has been supplied with the second solution, an anode is brought into contact with the sample reservoir 1, a cathode is brought into contact with the electrophoresis liquid reservoir 3, and a voltage is applied therebetween.
  • electroosmotic flow occurs in the capillary flow path 2
  • the second solution is introduced from the sample reservoir 1 into the capillary flow path 2
  • the second solution moves from the sample reservoir 1 toward the electrophoresis liquid reservoir 3, the component in the second solution is separated.
  • the applied voltage is preferably, for example, from 0.5 kV to 20 kV, more preferably from 0.5 kV to 10 kV, and still more preferably from 0.5 kV to 5 kV.
  • the determination method includes a detection process in which, for the aforementioned separated component, a value related to the component difference between the first solution and the second solution other than a value related to an analyte is optically detected.
  • the component separated in the capillary flow path 2 is measured by an optical method such as absorbance measurement.
  • the absorbance may be measured by irradiating light from the detection place 4.
  • the absorbance represents the absolute value of the common logarithm value of the ratio of the intensity of incident light to the intensity of transmitted light, and for example, an optical measurement value such as a value of the intensity of transmitted light itself which is simply detected without calculating the absorbance can be used for the determination method.
  • an optical measurement value such as a value of the intensity of transmitted light itself which is simply detected without calculating the absorbance can be used for the determination method.
  • explanation will be made taking as an example a case of calculating the absorbance.
  • the absorbance derived from the component difference between the first solution and the second solution other than the absorbance derived from an analyte is measured, and the absorbance derived from the component difference or the absorbance change amount obtained by using the component difference may be obtained.
  • the separated component may substantially not absorb light irradiated from the detection place 4, and for example, a change in apparent absorbance or absorbance change amount caused by a change in the intensity of transmitted light due to scattering, refraction, or the like caused by a difference in concentration of a specific substance may be detected.
  • the determination method includes a determination process in which whether the state of optical detection is favorable or poor is determined by comparing a value related to the optically detected component difference with a predetermined threshold.
  • the state of optical detection may be determined by comparing the absorbance or absorbance change amount derived from the component difference obtained in the detection process with a predetermined threshold value. For example, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value may indicate that the state of optical detection is favorable, and a value less than the threshold value may indicate that the state of optical detection is poor.
  • the determined state of optical detection may be displayed by display means such as a monitor (not shown), or may be notified by alarm means such as an alarm.
  • Such display, notification, or the like is preferably performed only when it is determined that the state of optical detection is poor. When it is determined that the state of optical detection is favorable, it is preferable that such display, notification, or the like is not performed.
  • the state of optical detection is poor, there is a conceivable influence of stray light or the like due to an optical fault of the analyzer, a production defect such as a scratch in a mechanism for removing stray light of a microchip, or foreign matter contamination in a mechanism for removing stray light of a microchip, and therefore, it is preferable to stop measurement of an analyte by the microchip.
  • the optically detected value related to the analyte may be corrected based on the value related to the above-described optically detected component difference (a correction process). For example, the absorbance or absorbance change amount derived from the aforementioned component difference obtained in the detection process is compared with the absorbance or absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or absorbance change amount derived from an analyte obtained in the detection process may be corrected according to the result of the comparison.
  • a correction process for example, the absorbance or absorbance change amount derived from the analyte, the accuracy of the measurement of the analyte can be enhanced.
  • the analysis method includes each process in the above-described determination method, and in the detection process, with respect to a separated component, a value related to an analyte is optically detected together with a value related to the component difference between the first solution and the second solution other than value related to the analyte. For example, in the analysis method, it is determined whether the state of optical detection is favorable or poor in the determination process and a value related to an analyte is optically detected in the detection process, that is, the absorbance or absorbance change amount which is an example of an optical measurement value or an optical measurement value change amount derived from an analyte is obtained.
  • the analyte contained in the second solution is hemoglobin, for example, it is preferable to measure the absorbance at a wavelength of 415 nm.
  • the component ratio or the like in the second solution may be obtained by calculating the peak height, the area of the peak, or the like of the electropherogram obtained by measuring the absorbance.
  • the microchip used in the embodiments of the invention may be a kit in combination with a cartridge for storing a solution for analysis.
  • the kit may include, for example, a microchip and cartridges storing a diluent and a first solution, respectively.
  • An analysis system includes: an arrangement unit to which a microchip including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side is fitted; separation means in which, in the microchip which is fitted to the arrangement unit and in which the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path; detection means for optically detecting a value related to the component difference between the first solution and the second solution for the separated component; and determination means for determining whether the state of the optical detection is favorable or poor by comparing, among values detected by the detection means, a value related to the component difference, other than a value related to the analyte, with a predetermined threshold value.
  • the analysis system includes an arrangement unit to which the microchip described above is fitted.
  • the microchip fitted to the arrangement unit includes, for example, a sample reservoir, an electrophoresis liquid reservoir, a capillary flow path connected to the sample reservoir and the electrophoresis liquid reservoir, and the like.
  • the electrophoresis liquid reservoir is supplied with a first solution for electrophoresis, and the capillary flow path is filled with the first solution by pressurization.
  • the sample reservoir is supplied with a second solution (for example, a solution obtained by diluting a sample containing an analyte) including an analyte, and by applying a voltage to the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path, whereby the aforementioned component is separated in the capillary flow path.
  • a second solution for example, a solution obtained by diluting a sample containing an analyte
  • analyte an analyte
  • the analysis system includes separation means for separating a component contained in the second solution in the capillary flow path by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution.
  • the separation means applies a predetermined voltage to an inside of the capillary flow path, and examples thereof include an anode to be inserted into the sample reservoir, a cathode to be inserted into the electrophoresis liquid reservoir, and voltage application means for applying a voltage to the anode and the cathode.
  • the analysis system includes detection means in which, for the aforementioned separated component, a value related to the component difference between the first solution and the second solution, preferably the difference in concentration of a specific substance between the first solution and the second solution, is optically detected.
  • detection means include a light emitting unit and a measurement unit.
  • the light emitting unit emits light for measuring the absorbance and is a unit that irradiates the detection place of a microchip with light.
  • the light emitting unit includes, for example, an LED chip that emits light in a predetermined wavelength range, an optical filter, a lens, and the like.
  • the light emitting unit may have a slit.
  • the measurement unit is a portion that receives light irradiated to the detection place of the microchip and measures the absorbance.
  • the measurement unit includes, for example, a photodiode, a photo IC, and the like.
  • the analysis system includes determination means for determining whether the state of optical detection is favorable or poor by comparing, among values related to component difference detected by the detection means, a value related to component difference, other than a value related to an analyte, with a predetermined threshold value. For example, when the value related to component difference detected by the detection means is the absorbance or absorbance change amount derived from a difference in concentration of a specific substance (excluding an analyte such as a biologically derived substance) contained in at least one of the first solution or the second solution, the determination means may determine whether the state of optical detection is favorable or poor by comparing the aforementioned absorbance or absorbance change amount with a predetermined threshold value.
  • an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value may be determined that the state of optical detection is favorable, and a value less than the threshold value may be determined that the state of optical detection is poor.
  • the analysis system may include display means such as a monitor for displaying a determined state of optical detection or may include notification means such as an alarm for notifying a determined state of optical detection. It is preferable that the display means and the notification means respectively display and notify only when the state of optical detection is determined to be poor, and it is preferable not to display and notify each when the state of optical detection is determined to be favorable.
  • the determination means When the state of optical detection is determined to be poor, the determination means preferably stops the measurement of an analyte by a microchip.
  • the determination means may include correction means in which, when it is determined that the state of optical detection is poor, the optically detected value related to the analyte is corrected based on the value related to the optically detected component difference other than value related to the analyte.
  • the absorbance or absorbance change amount derived from the aforementioned difference in concentration detected in the detection means is compared with the absorbance or absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or absorbance change amount derived from an analyte detected in the detection means may be corrected according to the result of the comparison.
  • the analysis system may include a diluent tank, an electrophoresis liquid tank, a pump, a control unit, and the like.
  • the diluent tank is, for example, a tank for storing a diluent for diluting a sample containing an analyte.
  • a solution (second solution) obtained by diluting the sample including the analyte may be supplied to the sample reservoir.
  • a microchip used in the analysis system may include a mixing tank for mixing the diluent supplied from the diluent tank and the sample containing the analyte.
  • the electrophoresis liquid tank is, for example, a tank for storing a first solution for electrophoresis supplied to the electrophoresis liquid reservoir.
  • the pump is, for example, a portion for supplying a diluent to a mixing tank by pressure application, supplying a first solution to an electrophoresis liquid reservoir by pressure application, and filling the first solution into a capillary flow path.
  • a solution (second solution) obtained by diluting a sample containing an analyte in a mixing tank may be supplied to a sample reservoir by a pump.
  • the second solution supplied to the sample reservoir may be made to flow by performing at least one of discharge and suction using a pump once or by repeating discharge and suction using a pump.
  • Fig. 2 shows a microchip provided with a mixing tank and having a structure capable of allowing a second solution stored in a sample reservoir to flow.
  • the microchip 200 shown in Fig. 2 includes a mixing tank 5 for mixing a diluent with a sample containing an analyte and a sample reservoir 11 including two openings and capable of allowing the second solution to flow.
  • a mixing tank 5 for mixing a diluent with a sample containing an analyte
  • a sample reservoir 11 including two openings and capable of allowing the second solution to flow.
  • at least one of discharge and suction using a pump may be performed once to cause the second solution stored in the sample reservoir 11 to flow, and alternatively, by repeatedly performing discharge and suction using a pump, the second solution stored in the sample reservoir 11 may be reciprocated in the y direction in Fig. 2 .
  • Example of a method of generating a shear flow at a connecting portion between the capillary flow path and the sample reservoir include a method in which the first solution is filled in the capillary flow path and the second solution is stored in the sample reservoir 11 in a state where a wall is provided in the connecting portion described above and then the wall is removed.
  • the pump is connected to the sample reservoir 11 of the microchip 200 to flow the second solution stored in the sample reservoir 11. This makes it easier for the pressure during pump operation to be stabilized.
  • a control unit controls each of the above-described components in the analysis system, and includes, for example, a CPU, a memory, an interface, and the like.
  • the control unit may also serve as a determination unit for determining whether optical detection is favorable or poor.
  • FIG. 3 is a sectional view showing a schematic configuration of an analysis system of one embodiment.
  • An analysis system 300 shown in Fig. 3 includes an arrangement unit 12 to which the microchip 200 is fixed, an anode 6, a cathode 7, a control unit 10, a photodiode 13, an LED chip 14, an optical filter 15, a lens 16, and a slit 17.
  • the anode 6 is inserted into the sample reservoir 11, and the cathode 7 is inserted into the electrophoresis liquid reservoir 12.
  • the LED chip 14 irradiates a detection place of the microchip 200 with light, and the photodiode 13 receives the light irradiated to the detection place of the microchip 200, and measures the absorbance.
  • the control unit 10 controls each component in the analysis system 300, and for example, the control unit 10 may perform control of a voltage applied to the anode 6 and the cathode 7, control of light emitted from the LED chip 14, measurement of absorbance based on light received by the photodiode 13, quality determination of optical detection, and the like.
  • the control unit 10 may perform control of each configuration not described in Fig. 3 such as control of pump discharge and suction, or control of supply, flow, and the like of the diluent, the first solution, the second solution, and the like.
  • each substance was added to distilled water to prepare the following electrophoresis liquid (1) and electrophoresis liquid (2).
  • ADAMS A1c Control Level 2 (manufactured by ARKRAY, Inc.) was dissolved in 300 ⁇ L of purified water to prepare a sample.
  • Peak 1 and Peak 2 represent the change in absorbance caused by the component difference between the electrophoresis liquid (1) and the electrophoresis liquid (2), and more specifically, Peak 1 is the absorbance change amount derived from NDSB-201, and Peak 2 is the absorbance change amount derived from EMULGEN LS-110.
  • the stray light ratio of the microchip (stray light ratio of the detection place) was measured.
  • the stray light ratio was measured using the four microchips after conducting the capillary electrophoresis described above.
  • a threshold value is predetermined and compared with Peak top value obtained by conducting capillary electrophoresis, and when the value obtained by conducting electrophoresis is equal to or more than the threshold value, it may be determined that the state of optical detection is favorable, and when the value obtained by conducting electrophoresis is less than the threshold value, it may be determined that the state of optical detection is poor.
  • the threshold value may be appropriately determined according to an allowable stray light ratio, an allowable deviation of HbA1c measurement value, and the like. Even when an optical measurement value of another index such as a value of the transmitted light intensity per se is used, determination can be done by setting a threshold value in the same way.

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Abstract

A determination method includes: using a microchip (100), including a capillary flow path (2) and a sample reservoir (1) connected to the capillary flow path (2) at an upstream side, to fill the capillary flow path (2) with a first solution for electrophoresis, and supply the sample reservoir (1) with a second solution containing an analyte; applying a voltage between the sample reservoir (1) supplied with the second solution and the inside of the capillary flow path (2) filled with the first solution, to move a component contained in the second solution in the capillary flow path (2) and separate the component in the capillary flow path (2); optically detecting a value related to a component difference between the first solution and the second solution, other than a value related to the analyte, for the separated component; and determining whether the optical detection is favorable or poor by comparing the optically detected value with a predetermined threshold value.

Description

    Technical Field
  • The present application relates to a determination method, an analysis method, and an analysis system.
    • Background Art
  • In the field of clinical tests, specimen analysis by capillary electrophoresis has conventionally been conducted. In recent years, specimen analysis by electrophoresis using a microchip provided with a capillary flow path has been conducted in order to miniaturize and simplify an apparatus.
  • For example, an analyzer for analyzing blood proteins such as hemoglobin by capillary electrophoresis has been proposed (see, for example, Patent Document 1). An analysis method for analyzing HbA1c which is a glycated hemoglobin using an electrophoresis chip has also been proposed (see, for example, Patent Document 2).
  • In capillary electrophoresis, a capillary is irradiated with light, light transmitted through the capillary is detected, and a sample moving in the capillary is analyzed. When analyzing a sample using such an optical method, in order to improve the analysis accuracy, it is necessary to remove internal stray light (extra light not contributing to measurement) due to aberration, irregular reflection, or the like of lenses constituting an optical system. Therefore, for the purpose of improving the analysis accuracy, a microchip having a function of reducing the influence of stray light has been proposed (see, for example, Patent Document 3).
    • RELATED ART DOCUMENTS Patent Documents
    • Patent Document 1 Japanese Patent Application Laid-Open ( JP-A) No. 2014-145775
    • Patent Document 2 Japanese Patent No. 5064497
    • Patent Document 3 Japanese Patent No. 5238028
    SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION
  • When the analyzer described in Patent Document 1 is used, there is a problem that while the analysis accuracy of blood protein is excellent, the device is large, operation is complicated, and high cost is incurred.
  • In the analysis method described in Patent Document 2, glycated hemoglobin can be measured using a rapid and inexpensive microchip. However, when stray light in optical detection exerts a large influence on the measurement accuracy, and a function of removing stray light is provided in an analyzer, the configuration of the analyzer becomes complicated and expensive.
  • Although the microchip described in Patent Document 3 has a function of reducing the influence of stray light, which enables analysis at a low cost with a simple device, in some cases, an influence of stray light is not reduced due to an optical fault of the analyzer, a production defect such as a scratch in a mechanism for removing stray light of a microchip, or foreign matter contamination in a mechanism for removing stray light of a microchip.
  • At least preferred embodiments of the present invention may provide a determination method, an analysis method, and an analysis system that can achieve high measurement accuracy easily and inexpensively.
    • MEANS FOR SOLVING THE PROBLEMS
  • A first aspect of the invention provides a determination method for determining whether the state of optical detection of a microchip is favorable or poor, comprising:
    • a supply process in which, by using the microchip including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side, the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte in a sample;
    • a separation process in which, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path;
    • a detection process for optically detecting a value related to a component difference between the first solution and the second solution, other than a value related to the analyte, for the separated component; and
    • a determination process for determining whether the state of the optical detection is favorable or poor by comparing the optically detected value related to the component difference with a predetermined threshold value.
  • Preferably, at least one of the first solution or the second solution contains a specific substance, and the component difference between the first solution and the second solution is a difference in concentration of the specific substance between the first solution and the second solution.
  • Preferably, the specific substance is an electrically neutral substance.
  • The specific substance may be at least one of 1-(3-sulfopropyl) pyridinium hydroxide inner salt or polyoxyalkylene alkyl ether.
  • Preferably, the optically detected value related to the component difference is an absorbance or an absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution, and in the determination process, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value is determined that the state of the optical detection is favorable, and a value less than the threshold value is determined that the state of the optical detection is poor.
  • Preferably, a shear flow is generated at a connecting portion between the capillary flow path and the sample reservoir in the supply process.
  • An analysis method may comprise the determination method as set out above, and in the detection process, with respect to the separated component, a value related to the analyte is optically detected together with the value related to the component difference between the first solution and the second solution other than the value related to the analyte.
  • Preferably, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference.
  • Preferably, at least one of the first solution or the second solution contains a specific substance, and the optically detected value related to the component difference is an absorbance or an absorbance change amount derived from a difference in concentration of the specific substance between the first solution and the second solution, and the value related to the analyte is an absorbance or an absorbance change amount derived from the analyte, and the absorbance or the absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution is compared with an absorbance or an absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or the absorbance change amount derived from the analyte is corrected according to the result of the comparison.
  • The analyte may be a biologically derived substance.
  • Optionally, the microchip is not reused.
  • A second aspect of the invention provides an analysis system for determining whether the state of optical detection of a microchip is favorable or poor, comprising:
    • an arrangement unit to which the microchip, including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side, is fitted;
    • separation means in which, in the microchip which is fitted to the arrangement unit and in which the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte in a sample, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path;
    • detection means for optically detecting a value related to a component difference between the first solution and the second solution for the separated component; and
    • determination means for determining whether the state of the optical detection is favorable or poor by comparing, among values detected by the detection means, a value related to the component difference, other than a value related to the analyte, with a predetermined threshold value.
  • Preferably, the value related to a component difference is an absorbance or an absorbance change amount derived from a difference in concentration of the specific substance between the first solution and the second solution, and the value related to the analyte is an absorbance or an absorbance change amount derived from the analyte, and in the determination means, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value is determined that the state of the optical detection is favorable, and a value less than the threshold value is determined that the state of the optical detection is poor.
  • Preferably, the determination means comprise correction means for correcting, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference, other than the value related to the analyte.
  • Preferably, the determination means comprise correction means for correcting, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference, other than the value related to the analyte, and in the correction means, the absorbance or the absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution is compared with an absorbance or an absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or the absorbance change amount derived from the analyte is corrected according to the result of the comparison.
    • EFFECTS OF THE INVENTION
  • At least preferred embodiments of the present invention may provide a determination method, an analysis method, and an analysis system that can achieve high measurement accuracy easily and inexpensively can be provided.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • Certain preferred embodiments of the present invention will now be described in greater detail by way of example only and with reference to the accompanying drawings in which:
    • Fig. 1 is a schematic configuration diagram of a microchip used in a determination method, an analysis method, and an analysis system of one embodiment, and Fig. 1A is a top view, and Fig. 1B is a sectional view taken along line C'-C' of Fig 1A;
    • Fig. 2 is a schematic configuration diagram of a microchip used in the analysis system of one embodiment, and Fig. 2A is a top view, and Fig. 2B is a sectional view taken along the line III - III in Fig. 2A, and Fig. 2C is a sectional view taken along line IV - IV of Fig. 2A;
    • Fig. 3 is a sectional view showing a schematic configuration of an analysis system of one embodiment; and
    • Figs. 4a to Fig. 4d are graphs showing results of electrophoresis using a microchip.
    MODE FOR CARRYING OUT THE INVENTION
  • Hereinafter, a determination method, an analysis method, and an analysis system will be described.
  • Herein, the numerical range expressed by using "to" means a range including numerical values described before and after "to" as a lower limit value and an upper limit value.
    • [Determination Method]
  • A determination method according to one aspect includes: a supply process in which, by using a microchip including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side, the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte; a separation process in which, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path; a detection process for optically detecting a value related to a component difference between the first solution and the second solution (an optical value related to component difference) other than a value related to the analyte (an optical value related to the analyte) for the separated component; and a determination process for determining whether the state of the optical detection is favorable or poor by comparing the optically detected value related to the component difference with a predetermined threshold value.
  • In the determination method, a value related to a component difference between the first solution and the second solution other than a value related to an analyte is optically detected for the component separated in the capillary flow path by electrophoresis. Then, by comparing the optically detected value related to the component difference with a predetermined threshold value, whether the state of optical detection is favorable or poor is determined. As a result, it is possible to determine whether the state of optical detection of a microchip is favorable or poor, and it is possible to easily and inexpensively achieve high measurement accuracy even when a microchip including a capillary flow path is used.
  • For example, when a value for optically detected component difference is an absorbance or an absorbance change amount derived from a difference in concentration of a specific substance (excluding an analyte such as a biologically derived substance) contained in at least one of the first solution or the second solution, during the determination process, the above-described absorbance or absorbance change amount may be compared with a predetermined threshold value to determine whether the state of optical detection is favorable or poor.
  • For example, the numerical values of an absorbance and absorbance change amount derived from a difference in concentration of a specific substance (excluding an analyte such as a biologically derived substance) contained in at least one of the first solution or the second solution tend to decrease as a stray light ratio increases, or the state of optical detection deteriorates. Even when optical detection is performed using a plurality of microchips using the first solution and the second solution having the same composition, results of the absorbance and absorbance change amount described above vary depending on the states of the microchips, for example, a production defect such as a scratch in a mechanism for removing stray light of a microchip, or a foreign matter contamination in a mechanism for removing stray light of a microchip. Accordingly, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value indicates that the state of optical detection is favorable (i.e. a value equal to or larger than the threshold value leads to the determination that the state of optical detection is favorable), and a value less than the threshold value indicates that the state of optical detection is poor (i.e. a value less than the threshold value leads to the determination that the state of optical detection is poor).
  • In the determination method, a value related to the component difference between the first solution and the second solution other than a value related to an analyte is optically detected, and whether the state of the optical detection is favorable or poor is determined by using a value related to such component difference.
  • Here, rather than determining whether the state of optical detection is favorable or poor by using a value related to an analyte, it is preferable to determine whether the state of optical detection is favorable or poor by using a value related to the component difference between the first solution and the second solution other than a value related to an analyte.
  • In the former case (i.e. when determining whether the state of optical detection is favorable or poor by using a value related to an analyte), when the concentration of a sample including an analyte is unknown and when a value related to the analyte such as the absorbance or absorbance change amount described above is different from a predetermined threshold value, it is impossible to determine whether the difference is due to the influence of stray light or due to the influence of the concentration of the sample including the analyte.
  • On the other hand, in the latter case, (i.e. when determining whether the state of optical detection is favorable or poor by using a value related to the component difference between the first solution and the second solution other than a value related to an analyte) it is possible to determine whether the state of optical detection is favorable or poor by a single operation by comparing a value related to the obtained component difference with a predetermined threshold value, regardless of the concentration of a sample including an analyte.
  • A microchip used in the determination method is used for a variety of analysis methods for a variety of samples, and is preferably used for analysis of substances (preferably biologically derived substances) in a sample by electrophoresis (preferably capillary electrophoresis). Examples of samples include a specimen derived from a living organism, a specimen derived from the environment, a metal, a chemical substance, and a pharmaceutical. The specimen derived from a living organism is not particularly limited, and examples thereof include urine, blood, hair, saliva, sweat, and nail. Examples of the blood specimen include a red blood cell, whole blood, serum, and plasma. Examples of the living body include a human, a non-human animal, and a plant, and examples of the non-human animal include a mammal other than humans, an amphibian reptile, a fish, a shellfish, and an insect. The specimen derived from the environment is not particularly limited, and examples thereof include food, water, soil, atmosphere, and air. Examples of the food include a fresh food, and a processed food. Examples of the water include drinking water, groundwater, river water, sea water, and domestic wastewater. The sample is preferably blood collected from a human body or the like. As the analyte, a biologically derived substance contained in a sample is preferable, and the biologically derived substance contained in blood is more preferable. Hemoglobin is also preferable among biological substances.
  • A diluted solution prepared, for example, by suspending, dispersing, or dissolving a solid including an analyte and a liquid containing an analyte (stock solution) in a liquid medium may be used as a sample. Examples of the liquid medium include water, and a buffer solution.
  • Examples of an analyte included in blood include hemoglobin (Hb), albumin (Alb), globulin (α1, α2, β, γ globulin), and fibrinogen. Examples of hemoglobin include normal hemoglobin (HbA0), glycated hemoglobin, modified hemoglobin, fetal hemoglobin (HbF), and mutated hemoglobin. Examples of glycated hemoglobin include hemoglobin Ala (HbA1a), hemoglobin A1b (HbA1b), hemoglobin A1c (HbA1c), and GHbLys. Examples of hemoglobin A1c include stable HbA1c (S-HbA1c), and unstable HbA1c (L-HbA1c). Examples of modified hemoglobin include carbamylated Hb, and acetylated Hb. Examples of the mutated hemoglobin include hemoglobin C (HbC), hemoglobin D (HbD), hemoglobin E (HbE), and hemoglobin S (HbS).
  • In the determination method, at least one of the first solution or the second solution contains a specific substance, and the component difference between the first solution and the second solution is preferably the difference in concentration of a specific substance between the first solution and the second solution. The first solution and the second solution preferably contain a specific substance, and more preferably, only the second solution contains a specific substance.
  • When a specific substance is contained only in the first solution or a specific substance is contained only in the second solution, there is a difference in concentration of a specific substance between the first solution and the second solution.
  • From the viewpoint of suppressing adverse effects on the electrophoresis of the first solution and the second solution, the specific substance is preferably an electrically neutral substance. Examples of the electrically neutral substance include a substance that is partially charged and is neutral as a whole, and a substance that does not have partial charges and is neutral, and specific examples thereof include an amphoteric substance and a nonionic substance. The specific substance may be used singly or two or more kinds thereof may be used. When two or more kinds of the specific substances are used, all the specific substances may be contained in the first solution and the second solution, respectively, or at least one of the specific substances may be contained in only one of the first solution and the second solution.
  • Examples of the amphoteric substance include phosphobetaine, sulfobetaine, and carbobetaine. More specifically, 1-(3-sulfopropyl) pyridinium hydroxide inner salt is preferable.
  • Examples of the nonionic substance include a sugar such as glucose or starch, urea, an alcohol such as polyethylene glycol, a nonionic surfactant, and a nonionic polymer. As the nonionic substance, polyoxyalkylene alkyl ether is preferable.
  • The second solution preferably contains an electrically neutral substance. As a result, an electrically neutral substance contained in the second solution is subjected to an electroosmotic flow, and is electrophoresed at the interface between the first solution and the second solution in the capillary flow path, whereby whether the state of optical detection is favorable or poor is determined without being affected by measurement of an analyte.
  • The microchip used in the determination method will be described with reference to Fig. 1. Fig. 1 is a schematic configuration diagram of a microchip used in a determination method and an analysis method of one embodiment, and Fig. 1A is a top view, and Fig. 1B is a sectional view taken along line C'-C' of Fig 1A. As shown in Fig. 1, a microchip 100 includes a sample reservoir 1, an electrophoresis liquid reservoir 3, a capillary flow path 2, and a detector 4. The microchip 100 may be manufactured, for example, by joining a pair of base materials, which are substantially rectangular plate-shaped members. More specifically, the microchip 100 may be manufactured by joining a base material having through holes corresponding to the sample reservoir 1 and the electrophoresis liquid reservoir 3 and a base material including a fine groove corresponding to the capillary flow path 2 in such a manner that both ends of the groove face portions of the two through holes respectively.
  • Examples of the material of the base material constituting the microchip include glass, fused silica, and a resin, and from the viewpoints of cost, ease of processing, and ease of immobilization of a cationic polymer, a resin is preferable. Examples of the resin include an acrylic resin such as polymethyl methacrylate (PMMA), polymethyl methacrylate, polycarbonate, polyvinylidene chloride, cyclic polyolefin, polyether ether ketone, polystyrene, polytetrafluoroethylene (PTFE), cycloolefin, polypropylene, and polyethylene, and from the viewpoint of excellent light transmittance, methyl polymethacrylate is preferable.
  • As the microchip used in the determination method and the analysis method described below, a disposable type analysis chip not for reusing may be used.
  • The sample reservoir 1 is a tank that is supplied with a second solution containing an analyte such as a biologically derived substance from an opening of the sample reservoir 1 and stores the supplied second solution. The sample reservoir 1 is connected to the end of the capillary flow path 2.
  • The main agent of a diluent is not particularly limited, and examples thereof include water, and physiological saline, and a substance which can be contained in the first solution for electrophoresis described below as a preferred example may be added to the diluent. For example, the diluent may be one obtained by adding a compound including a cathodic group to the main agent. Examples of the compound including a cathodic group include a cathodic group-containing polysaccharide, and more specific examples thereof include a sulfated polysaccharide, a carboxylated polysaccharide, a sulfonated polysaccharide, and a phosphorylated polysaccharide. As the carboxylated polysaccharide, alginic acid and a salt thereof (for example, sodium alginate) is preferable. As the sulfated polysaccharide, for example, chondroitin sulfate is preferable. There are seven kinds of chondroitin sulfuric acid, A, B, C, D, E, H, and K, and any of them may be used. The concentration of a compound including a cathodic group (chondroitin sulfate) is preferably, for example, in the range of from 0.01% by mass to 5% by mass.
  • The electrophoresis liquid reservoir 3 is a reservoir for supplying a first solution for electrophoresis from an opening of the electrophoresis reservoir 3 and storing the supplied first solution. The electrophoresis liquid reservoir 3 is connected to an end of the capillary flow path 2, and the first solution is filled in the capillary flow path 2 by pressurization.
  • The first solution which is an electrophoresis liquid used for electrophoresis is a medium which is supplied from the opening to the electrophoresis liquid reservoir 3 and filled in the capillary flow path 2 to generate electroosmotic flow in electrophoresis. The first solution includes water, and/or physiological saline and the like. The first solution preferably contains an acid. Examples of the acid include citric acid, maleic acid, tartaric acid, succinic acid, fumaric acid, phthalic acid, malonic acid, and malic acid. The first solution preferably contains a weak base. Examples of the weak base include arginine, lysine, histidine, and a tris. The pH of the first solution is preferably, for example, in the range of pH from 4.5 to 6. Examples of the buffer of the first solution include MES, ADA, ACES, BES, MOPS, TES, and HEPES. The compound including a cathodic group described in the description of the diluent may also be added to the first solution. The concentration of the compound including a cathodic group (chondroitin sulfate or the like) is preferably, for example, in the range of from 0.01% by mass to 5% by mass.
  • The capillary flow path 2 is connected to the sample reservoir 1 and the electrophoresis liquid reservoir 3, and is a flow path for analyzing an analyte in the second solution supplied to the sample reservoir 1, and is preferably a capillary channel for analyzing an analyte by electrophoresis.
  • The size of the capillary flow path 2 is not particularly limited, and preferably, for example, the depth is from 25 µm to 100 µm, the width is from 25 µm to 100 µm, and the length is from 5 mm to 150 mm.
  • From the viewpoint of enhancing the analytical performance, the capillary flow path 2 may be provided with an electric charge, for example, a positive electric charge on a wall face. The method of imparting a positive charge is not particularly limited, and a cationic polymer may be immobilized in a region corresponding to the capillary flow path 2 in the above-described pair of base materials before joining the pair of base materials described above.
  • The detection place 4 is for entering and emitting light to be analyzed when conducting analysis by electrophoresis on the microchip 100, and is used, for example, for measuring absorbance. The detection place 4 is formed at a position facing at least a portion of the capillary flow path 2. The position of the detection place 4 may be appropriately determined according to the length of the capillary flow path 2 and the like.
    • (Supply Process)
  • Hereinafter, each process of the determination method using the above-described microchip 100 will be described. The determination method includes a supply process in which, by using the microchip 100 described above, the capillary flow path 2 is filled with the first solution and the sample reservoir 1 is supplied with the second solution containing an analyte. For example, the first solution stored in the electrophoresis liquid reservoir 3 may be pressurized to fill the capillary flow path 2 with the first solution, and as the second solution, one obtained by diluting a sample containing an analyte with the aforementioned diluent may be used.
    • (Separation Process)
  • Next, the determination method includes a separation process in which, by applying a voltage between the sample reservoir 1 supplied with the second solution and an inside of the capillary flow path 2 filled with the first solution, a component contained in the second solution moves in the capillary flow path 2 and the aforementioned component is separated in the capillary flow path 2.
  • In the microchip 100, when the electrophoresis liquid reservoir 3 has been supplied with the first solution, and the capillary flow path 2 has been filled with the first solution by pressurization, and the sample reservoir 1 has been supplied with the second solution, an anode is brought into contact with the sample reservoir 1, a cathode is brought into contact with the electrophoresis liquid reservoir 3, and a voltage is applied therebetween. As a result, electroosmotic flow occurs in the capillary flow path 2, the second solution is introduced from the sample reservoir 1 into the capillary flow path 2, and when the second solution moves from the sample reservoir 1 toward the electrophoresis liquid reservoir 3, the component in the second solution is separated. The applied voltage is preferably, for example, from 0.5 kV to 20 kV, more preferably from 0.5 kV to 10 kV, and still more preferably from 0.5 kV to 5 kV.
    • (Detection Process)
  • The determination method includes a detection process in which, for the aforementioned separated component, a value related to the component difference between the first solution and the second solution other than a value related to an analyte is optically detected. The component separated in the capillary flow path 2 is measured by an optical method such as absorbance measurement. For example, the absorbance may be measured by irradiating light from the detection place 4. The absorbance represents the absolute value of the common logarithm value of the ratio of the intensity of incident light to the intensity of transmitted light, and for example, an optical measurement value such as a value of the intensity of transmitted light itself which is simply detected without calculating the absorbance can be used for the determination method. In the following, explanation will be made taking as an example a case of calculating the absorbance. At this time, for the separated component, the absorbance derived from the component difference between the first solution and the second solution other than the absorbance derived from an analyte is measured, and the absorbance derived from the component difference or the absorbance change amount obtained by using the component difference may be obtained. The separated component may substantially not absorb light irradiated from the detection place 4, and for example, a change in apparent absorbance or absorbance change amount caused by a change in the intensity of transmitted light due to scattering, refraction, or the like caused by a difference in concentration of a specific substance may be detected.
    • (Determination Process)
  • The determination method includes a determination process in which whether the state of optical detection is favorable or poor is determined by comparing a value related to the optically detected component difference with a predetermined threshold. The state of optical detection may be determined by comparing the absorbance or absorbance change amount derived from the component difference obtained in the detection process with a predetermined threshold value. For example, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value may indicate that the state of optical detection is favorable, and a value less than the threshold value may indicate that the state of optical detection is poor. The determined state of optical detection may be displayed by display means such as a monitor (not shown), or may be notified by alarm means such as an alarm. Such display, notification, or the like is preferably performed only when it is determined that the state of optical detection is poor. When it is determined that the state of optical detection is favorable, it is preferable that such display, notification, or the like is not performed. When it is determined that the state of optical detection is poor, there is a conceivable influence of stray light or the like due to an optical fault of the analyzer, a production defect such as a scratch in a mechanism for removing stray light of a microchip, or foreign matter contamination in a mechanism for removing stray light of a microchip, and therefore, it is preferable to stop measurement of an analyte by the microchip. Besides, when it is determined that the state of optical detection is poor, the optically detected value related to the analyte may be corrected based on the value related to the above-described optically detected component difference (a correction process). For example, the absorbance or absorbance change amount derived from the aforementioned component difference obtained in the detection process is compared with the absorbance or absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or absorbance change amount derived from an analyte obtained in the detection process may be corrected according to the result of the comparison. By correcting a value related to an analyte, for example, the absorbance or absorbance change amount derived from the analyte, the accuracy of the measurement of the analyte can be enhanced.
    • [Analysis Method]
  • The analysis method includes each process in the above-described determination method, and in the detection process, with respect to a separated component, a value related to an analyte is optically detected together with a value related to the component difference between the first solution and the second solution other than value related to the analyte. For example, in the analysis method, it is determined whether the state of optical detection is favorable or poor in the determination process and a value related to an analyte is optically detected in the detection process, that is, the absorbance or absorbance change amount which is an example of an optical measurement value or an optical measurement value change amount derived from an analyte is obtained.
  • When the analyte contained in the second solution is hemoglobin, for example, it is preferable to measure the absorbance at a wavelength of 415 nm. The component ratio or the like in the second solution may be obtained by calculating the peak height, the area of the peak, or the like of the electropherogram obtained by measuring the absorbance.
    • [Kit]
  • The microchip used in the embodiments of the invention may be a kit in combination with a cartridge for storing a solution for analysis. The kit may include, for example, a microchip and cartridges storing a diluent and a first solution, respectively.
    • [Analysis System]
  • An analysis system includes: an arrangement unit to which a microchip including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side is fitted; separation means in which, in the microchip which is fitted to the arrangement unit and in which the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path; detection means for optically detecting a value related to the component difference between the first solution and the second solution for the separated component; and determination means for determining whether the state of the optical detection is favorable or poor by comparing, among values detected by the detection means, a value related to the component difference, other than a value related to the analyte, with a predetermined threshold value. With this analysis system, it is possible to determine whether the state of optical detection of the microchip is favorable or poor, and it is possible to easily and inexpensively achieve high measurement accuracy even when a microchip including a capillary flow path is used.
  • The analysis system includes an arrangement unit to which the microchip described above is fitted. The microchip fitted to the arrangement unit includes, for example, a sample reservoir, an electrophoresis liquid reservoir, a capillary flow path connected to the sample reservoir and the electrophoresis liquid reservoir, and the like. The electrophoresis liquid reservoir is supplied with a first solution for electrophoresis, and the capillary flow path is filled with the first solution by pressurization. The sample reservoir is supplied with a second solution (for example, a solution obtained by diluting a sample containing an analyte) including an analyte, and by applying a voltage to the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path, whereby the aforementioned component is separated in the capillary flow path.
  • The analysis system includes separation means for separating a component contained in the second solution in the capillary flow path by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution. The separation means applies a predetermined voltage to an inside of the capillary flow path, and examples thereof include an anode to be inserted into the sample reservoir, a cathode to be inserted into the electrophoresis liquid reservoir, and voltage application means for applying a voltage to the anode and the cathode.
  • The analysis system includes detection means in which, for the aforementioned separated component, a value related to the component difference between the first solution and the second solution, preferably the difference in concentration of a specific substance between the first solution and the second solution, is optically detected. Examples of the detection means include a light emitting unit and a measurement unit.
  • For example, the light emitting unit emits light for measuring the absorbance and is a unit that irradiates the detection place of a microchip with light. The light emitting unit includes, for example, an LED chip that emits light in a predetermined wavelength range, an optical filter, a lens, and the like. The light emitting unit may have a slit.
  • For example, the measurement unit is a portion that receives light irradiated to the detection place of the microchip and measures the absorbance. The measurement unit includes, for example, a photodiode, a photo IC, and the like.
  • The analysis system includes determination means for determining whether the state of optical detection is favorable or poor by comparing, among values related to component difference detected by the detection means, a value related to component difference, other than a value related to an analyte, with a predetermined threshold value. For example, when the value related to component difference detected by the detection means is the absorbance or absorbance change amount derived from a difference in concentration of a specific substance (excluding an analyte such as a biologically derived substance) contained in at least one of the first solution or the second solution, the determination means may determine whether the state of optical detection is favorable or poor by comparing the aforementioned absorbance or absorbance change amount with a predetermined threshold value. Specifically, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value may be determined that the state of optical detection is favorable, and a value less than the threshold value may be determined that the state of optical detection is poor. The analysis system may include display means such as a monitor for displaying a determined state of optical detection or may include notification means such as an alarm for notifying a determined state of optical detection. It is preferable that the display means and the notification means respectively display and notify only when the state of optical detection is determined to be poor, and it is preferable not to display and notify each when the state of optical detection is determined to be favorable. When the state of optical detection is determined to be poor, the determination means preferably stops the measurement of an analyte by a microchip. Besides, the determination means may include correction means in which, when it is determined that the state of optical detection is poor, the optically detected value related to the analyte is corrected based on the value related to the optically detected component difference other than value related to the analyte. For example, in the determination means, the absorbance or absorbance change amount derived from the aforementioned difference in concentration detected in the detection means is compared with the absorbance or absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or absorbance change amount derived from an analyte detected in the detection means may be corrected according to the result of the comparison.
  • Further, the analysis system may include a diluent tank, an electrophoresis liquid tank, a pump, a control unit, and the like.
  • The diluent tank is, for example, a tank for storing a diluent for diluting a sample containing an analyte. For example, after mixing a diluent supplied from the diluent tank and a sample containing an analyte in a mixing tank, a solution (second solution) obtained by diluting the sample including the analyte may be supplied to the sample reservoir. In this case, a microchip used in the analysis system may include a mixing tank for mixing the diluent supplied from the diluent tank and the sample containing the analyte.
  • The electrophoresis liquid tank is, for example, a tank for storing a first solution for electrophoresis supplied to the electrophoresis liquid reservoir.
  • The pump is, for example, a portion for supplying a diluent to a mixing tank by pressure application, supplying a first solution to an electrophoresis liquid reservoir by pressure application, and filling the first solution into a capillary flow path. A solution (second solution) obtained by diluting a sample containing an analyte in a mixing tank may be supplied to a sample reservoir by a pump. The second solution supplied to the sample reservoir may be made to flow by performing at least one of discharge and suction using a pump once or by repeating discharge and suction using a pump.
  • Fig. 2 shows a microchip provided with a mixing tank and having a structure capable of allowing a second solution stored in a sample reservoir to flow. The microchip 200 shown in Fig. 2 includes a mixing tank 5 for mixing a diluent with a sample containing an analyte and a sample reservoir 11 including two openings and capable of allowing the second solution to flow. When allowing the second solution stored in the sample reservoir 11 to flow, for example, at least one of discharge and suction using a pump may be performed once to cause the second solution stored in the sample reservoir 11 to flow, and alternatively, by repeatedly performing discharge and suction using a pump, the second solution stored in the sample reservoir 11 may be reciprocated in the y direction in Fig. 2. At this time, in the vicinity of a connecting portion between the sample reservoir 11 and the capillary flow path 2, a shear flow occurs because the first solution in the capillary flow path 2 hardly moves. As a result, a shear flow that generates a clear boundary between the first solution filled in the capillary flow path 2 and the second solution stored in the sample reservoir 11 is generated and the analysis accuracy tends to be improved.
  • Example of a method of generating a shear flow at a connecting portion between the capillary flow path and the sample reservoir include a method in which the first solution is filled in the capillary flow path and the second solution is stored in the sample reservoir 11 in a state where a wall is provided in the connecting portion described above and then the wall is removed.
  • In the case of allowing the second solution stored in the sample reservoir 11 to flow by repeating discharge and suction using the pump, it is preferable that, after the pump is strongly idled, the pump is connected to the sample reservoir 11 of the microchip 200 to flow the second solution stored in the sample reservoir 11. This makes it easier for the pressure during pump operation to be stabilized.
  • A control unit controls each of the above-described components in the analysis system, and includes, for example, a CPU, a memory, an interface, and the like. The control unit may also serve as a determination unit for determining whether optical detection is favorable or poor.
  • The analysis system will be described with reference to Fig. 3. Fig. 3 is a sectional view showing a schematic configuration of an analysis system of one embodiment. An analysis system 300 shown in Fig. 3 includes an arrangement unit 12 to which the microchip 200 is fixed, an anode 6, a cathode 7, a control unit 10, a photodiode 13, an LED chip 14, an optical filter 15, a lens 16, and a slit 17.
  • The anode 6 is inserted into the sample reservoir 11, and the cathode 7 is inserted into the electrophoresis liquid reservoir 12. The LED chip 14 irradiates a detection place of the microchip 200 with light, and the photodiode 13 receives the light irradiated to the detection place of the microchip 200, and measures the absorbance.
  • The control unit 10 controls each component in the analysis system 300, and for example, the control unit 10 may perform control of a voltage applied to the anode 6 and the cathode 7, control of light emitted from the LED chip 14, measurement of absorbance based on light received by the photodiode 13, quality determination of optical detection, and the like. The control unit 10 may perform control of each configuration not described in Fig. 3 such as control of pump discharge and suction, or control of supply, flow, and the like of the diluent, the first solution, the second solution, and the like.
  • Although the determination method, analysis method, and analysis system according to have been described above, the application is not limited to this disclosure and various improvements, changes, and modifications may be made based on knowledge of those skilled in the art without departing from the scope of the application, as defined by the appended claims. The features described as features of the determination method, analysis method, and analysis system may be combined as appropriate.
    • EXAMPLES
  • Next, one embodiment will be described based on the following Examples, but the application is not limited to the following Examples.
    • < Preparation of Microchip >
  • In this Example, a resin-made microchip having each configuration satisfying the following conditions was prepared. The inner wall of a capillary flow path is covered with polydiallyldimethylammonium chloride (weight average molecular weight of from 100,000 to 500,000).
    • Sample reservoir ••• Capacity 10 µL
    • Electrophoresis liquid reservoir ••• Capacity 10 µL
    • Capillary flow path ••• Depth 0.04 mm × width 0.04 mm × length 30 mm (Optical detection is performed by a detection place that is 20 mm away from the side of the sample reservoir)
    < Preparation of Electrophoresis Liquid >
  • First, each substance was added to distilled water to prepare the following electrophoresis liquid (1) and electrophoresis liquid (2).
    • (Electrophoresis Liquid (1))
  • 40 mM citric acid, 1.25% w/v chondroitin sulfate C sodium, 20 mM piperazine, 0.1% w/v polyoxyalkylene alkyl ether (trade name: EMULGEN LS-110, manufactured by Kao Corporation), 0.02% w/v sodium azide, 0.025% w/v proclin 300, dimethylaminoethanol (for pH adjustment), pH 5.0
    • (Electrophoresis Liquid (2))
  • 38 mM citric acid, 0.95% w/v chondroitin sulfate C sodium, 475 mM 1-(3-sulfopropyl) pyridinium hydroxide inner salt (NDSB-201), 19 mM morpholinoethanesulfonic acid (MES), 0.4% w/v polyoxyalkylene alkyl ether (trade name: EMULGEN LS-110, manufactured by Kao Corporation), 0.02% w/v sodium azide, 0.025% w/v proclin 300, dimethylaminoethanol (for pH adjustment), pH 6.0
    • < Preparation of Sample >
  • ADAMS A1c Control Level 2 (manufactured by ARKRAY, Inc.) was dissolved in 300 µL of purified water to prepare a sample.
    • < Conduct of Capillary Electrophoresis >
  • Analysis of hemoglobin in the sample was conducted according to the following procedure. Capillary electrophoresis was carried out using four microchips.
    1. 1) A microchip was set in a dedicated device manufactured by ARKRAY, Inc.
    2. 2) The following electrophoresis liquid (1) was added to the electrophoresis liquid tank (electrophoresis liquid reservoir) of the microchip, and the electrophoresis liquid (1) was filled in the capillary flow path by pressurization.
    3. 3) 1.5 µL of the sample was added to 60 µL of electrophoresis liquid (2) to obtain a diluted sample.
    4. 4) The diluted sample was added to a sample tank (sample reservoir).
    5. 5) An anode was brought into contact with the sample reservoir and a cathode was brought into contact with the electrophoresis liquid tank, whereby electrophoresis was started with a constant current control of 70 µA.
    6. 6) The optical absorbance at 415 nm was measured at a detection place to obtain an electropherogram. Electrophoresis was performed for 40 seconds.
  • The results of the capillary electrophoresis are as shown in Fig. 4a to Fig. 4d. In Fig. 4a to Fig. 4c, Peak 1 and Peak 2 represent the change in absorbance caused by the component difference between the electrophoresis liquid (1) and the electrophoresis liquid (2), and more specifically, Peak 1 is the absorbance change amount derived from NDSB-201, and Peak 2 is the absorbance change amount derived from EMULGEN LS-110.
    • < Stray Light Ratio Measurement >
  • According to the following procedure, the stray light ratio of the microchip (stray light ratio of the detection place) was measured. The stray light ratio was measured using the four microchips after conducting the capillary electrophoresis described above.
    1. 1) A microchip was set in a dedicated device manufactured by ARKRAY, Inc.
    2. 2) The optical absorbance at 415 nm was measured at the detection place (Signal).
    3. 3) A light shielding liquid was added to the electrophoresis liquid tank (electrophoresis liquid reservoir) of the microchip, and the light shielding liquid was filled in the capillary flow path by pressurization.
    4. 4) The absorbance at 415 nm was measured at the detection place (Background).
    5. 5) The stray light ratio was calculated by "(Signal/Background) × 100".
    < Optical Quality Determination >
  • Based on the electropherogram information shown in Fig. 4a to Fig. 4d, the top values of Peak 1 and Peak 2 and HbA1c measured values (NGSP%) were obtained. These values are shown in Table 1 together with the stray light ratio determined by the stray light ratio measurement described above. [Table 1]
    Microchip Stray light ratio (%) HbA1c (NGSP%) Peak 1 Top value Peak 2 Top value
    1 2 10.01 6.5 0.69
    2 18 10.26 3.9 0.39
    3 37 10.35 2.3 0.26
    4 51 10.65 0.0 0.00
  • As shown in Table 1, there was a tendency that the HbA1c measured value tended to increase as the stray light ratio was larger, and the top values of Peak 1 and Peak 2 tended to decrease. Therefore, for example, with respect to top value of at least one of Peak 1 and Peak 2, a threshold value is predetermined and compared with Peak top value obtained by conducting capillary electrophoresis, and when the value obtained by conducting electrophoresis is equal to or more than the threshold value, it may be determined that the state of optical detection is favorable, and when the value obtained by conducting electrophoresis is less than the threshold value, it may be determined that the state of optical detection is poor. The threshold value may be appropriately determined according to an allowable stray light ratio, an allowable deviation of HbA1c measurement value, and the like. Even when an optical measurement value of another index such as a value of the transmitted light intensity per se is used, determination can be done by setting a threshold value in the same way.
    • DESCRIPTION OF SYMBOLS
  • 1, 11 Sample reservoir, 2 Capillary flow path, 3 Electrophoresis liquid reservoir, 4 Detection place, 5 Mixing tank, 6 Anode, 7 Cathode, 10 Control unit, 12 Arrangement unit, 13 Photodiode (Measurement unit), 14 LED chip (light emitting unit), 15 Optical filter, 16 Lens, 17 Slit, 100, 200 Microchip, 300 Analysis system

Claims (15)

  1. A determination method for determining whether the state of optical detection of a microchip is favorable or poor, comprising:
    a supply process in which, by using the microchip including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side, the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte in a sample;
    a separation process in which, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path;
    a detection process for optically detecting a value related to a component difference between the first solution and the second solution, other than a value related to the analyte, for the separated component; and
    a determination process for determining whether the state of the optical detection is favorable or poor by comparing the optically detected value related to the component difference with a predetermined threshold value.
  2. The determination method according to claim 1, wherein:
    at least one of the first solution or the second solution contains a specific substance, and
    the component difference between the first solution and the second solution is a difference in concentration of the specific substance between the first solution and the second solution.
  3. The determination method according to claim 2, wherein the specific substance is an electrically neutral substance.
  4. The determination method according to claim 2 or 3, wherein the specific substance is at least one of 1-(3-sulfopropyl) pyridinium hydroxide inner salt or polyoxyalkylene alkyl ether.
  5. The determination method according to any one of claims 2 to 4, the optically detected value related to the component difference is an absorbance or an absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution, and
    in the determination process, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value is determined that the state of the optical detection is favorable, and a value less than the threshold value is determined that the state of the optical detection is poor.
  6. The determination method according to any one of claims 1 to 5, wherein a shear flow is generated at a connecting portion between the capillary flow path and the sample reservoir in the supply process.
  7. An analysis method, comprising each process of the determination method according to any one of claims 1 to 6, wherein, in the detection process, with respect to the separated component, a value related to the analyte is optically detected together with the value related to the component difference between the first solution and the second solution other than the value related to the analyte.
  8. The analysis method according to claim 7, wherein when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference.
  9. The analysis method according to claim 7, wherein at least one of the first solution or the second solution contains a specific substance, and the optically detected value related to the component difference is an absorbance or an absorbance change amount derived from a difference in concentration of the specific substance between the first solution and the second solution, and the value related to the analyte is an absorbance or an absorbance change amount derived from the analyte, and
    the absorbance or the absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution is compared with an absorbance or an absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or the absorbance change amount derived from the analyte is corrected according to the result of the comparison.
  10. The analysis method according to any one of claims 7 to 9, wherein the analyte is a biologically derived substance.
  11. The analysis method according to any one of claims 7 to 10, wherein the microchip is not reused.
  12. An analysis system for determining whether the state of optical detection of a microchip is favorable or poor, comprising:
    an arrangement unit to which the microchip, including a capillary flow path and a sample reservoir connected to the capillary flow path at an upstream side, is fitted;
    separation means in which, in the microchip which is fitted to the arrangement unit and in which the capillary flow path is filled with a first solution for electrophoresis, and the sample reservoir is supplied with a second solution containing an analyte in a sample, by applying a voltage between the sample reservoir supplied with the second solution and an inside of the capillary flow path filled with the first solution, a component contained in the second solution moves in the capillary flow path and the component is separated in the capillary flow path;
    detection means for optically detecting a value related to a component difference between the first solution and the second solution for the separated component; and
    determination means for determining whether the state of the optical detection is favorable or poor by comparing, among values detected by the detection means, a value related to the component difference, other than a value related to the analyte, with a predetermined threshold value.
  13. The analysis system according to claim 12, wherein the value related to a component difference is an absorbance or an absorbance change amount derived from a difference in concentration of the specific substance between the first solution and the second solution, and the value related to the analyte is an absorbance or an absorbance change amount derived from the analyte, and
    in the determination means, an absorbance or an absorbance change amount corresponding to an allowable stray light ratio is set as a threshold value, and a value equal to or larger than the threshold value is determined that the state of the optical detection is favorable, and a value less than the threshold value is determined that the state of the optical detection is poor.
  14. The analysis system according to claim 12 or 13, wherein the determination means comprise correction means for correcting, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference, other than the value related to the analyte.
  15. The analysis system according to claim 13, wherein the determination means comprise correction means for correcting, when it is determined that the state of the optical detection is poor, the value related to the analyte is corrected based on the optically detected value related to the component difference, other than the value related to the analyte, and
    in the correction means, the absorbance or the absorbance change amount derived from the difference in concentration of the specific substance between the first solution and the second solution is compared with an absorbance or an absorbance change amount corresponding to a predetermined specific stray light ratio, and the absorbance or the absorbance change amount derived from the analyte is corrected according to the result of the comparison.
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US10948399B2 (en) 2021-03-16

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